The present invention relates to medicaments that are useful for modulating lipoprotein levels in vivo. More particularly, the invention relates to medicaments that modify the activity of the Lipolysis Stimulated Receptor (LSR) and that can be used to influence the partitioning of dietary lipids between the liver and peripheral tissues, including adipose tissue.
Obesity is a public health problem which is both serious and widespread. One-third of the population in industrialized countries has an excess weight of at least 20% relative to the ideal weight. The phenomenon continues to worsen, particularly in regions of the globe where economies are modernizing. In the United States, the number of obese people has escalated from 25% at the end of the 70s to 33% at the beginning of the 90s.
Obesity considerably increases the risk of developing cardiovascular or metabolic diseases. It is estimated that if the entire population had an ideal weight, the risk of coronary insufficiency would decrease by 25% and that of cardiac insufficiency and of cerebral vascular accidents by 35%. Coronary insufficiency, atheromatous disease and cardiac insufficiency are at the forefront of the cardiovascular complications induced by obesity. For an excess weight greater than 30%, the incidence of coronary diseases is doubled in subjects under 50 years. Studies carried out for other diseases are equally eloquent. For an excess weight of 20%, the risk of high blood pressure is doubled. For an excess weight of 30%, the risk of developing a non-insulin-dependent diabetes is tripled. That of hyperlipidemias is multiplied six fold.
The list of diseases having onsets promoted by obesity is long: hyperuricemia (11.4% in obese subjects, against 3.4% in the general population), digestive pathologies, abnormalities in hepatic functions, and even certain cancers.
Whether the physiological changes in obesity are characterized by an increase in the number of adipose cells, or by an increase in the quantity of triglycerides stored in each adipose cell, or by both, this excess weight results mainly from an imbalance between the quantities of calories consumed and those of the calories used by the body. Studies on the causes of this imbalance have been in several directions. Some have focused on studying the mechanism of absorption of foods, and therefore the molecules which control food intake and the feeling of satiety. Other studies have characterized the pathways through which the body uses its calories.
The treatments for obesity which have been proposed are of four types. Food restriction is the most frequently used. The obese individuals are advised to change their dietary habits so as to consume fewer calories. This type of treatment is effective in the short-term. However, the recidivation rate is very high. The increase in calorie use through physical exercise is also proposed. This treatment is ineffective when applied alone, but it improves, however, weight-loss in subjects on a low-calorie diet. Gastrointestinal surgery, which reduces the absorption of the calories ingested, is effective but has been virtually abandoned because of the side effects which it causes. The medicinal approach uses either the anorexigenic action of molecules involved at the level of the central nervous system, or the effect of molecules which increase energy use by increasing the production of heat. The prototypes of this type of molecule are the thyroid hormones which uncouple oxidative phosphorylations of the mitochondrial respiratory chain. The side effects and the toxicity of this type of treatment make their use dangerous. An approach which aims to reduce the absorption of dietary lipids by sequestering them in the lumen of the digestive tube is also in place. However, it induces physiological imbalances which are difficult to tolerate: deficiency in the absorption of fat soluble vitamins, flatulence and steatorrhoea. Whatever the envisaged therapeutic approach, the treatments of obesity are all characterized by an extremely high recidivation rate.
The molecular mechanisms responsible for obesity in man are complex and involve genetic and environmental factors. Because of the low efficiency of the treatments known up until now, it is urgent to define the genetic mechanisms which determine obesity, so as to be able to develop better targeted medicaments.
More than 20 genes have been studied as possible candidates, either because they have been implicated in diseases of which obesity is one of the clinical manifestations, or because they are homologues of genes involved in obesity in animal models. Situated in the 7q31 chromosomal region, the OB gene is one of the most widely studied. Its product, leptin, is involved in the mechanisms of satiety. Leptin is a plasma protein of 16 kDa produced by the adipocytes under the action of various stimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptin gene; this protein is undetectable in the plasma of these animals. The administration of leptin obtained by genetic engineering to ob/ob mice corrects their relative hyperphagia and allows normalization of their weight. This anorexigenic effect of leptin calls into play a receptor of the central nervous system: the ob receptor which belongs to the family of class 1 cytokine receptors. The ob receptor is deficient in obese mice of the db/db strain. The administration of leptin to these mice has no effect on their food intake and does not allow substantial reduction in their weight. The mechanisms by which the ob receptors transmit the signal for satiety are not precisely known. It is possible that neuropeptide Y is involved in this signalling pathway. It is important to specify at this stage that the ob receptors are not the only regulators of appetite. The Melanocortin 4 receptor is also involved since mice made deficient in this receptor are obese (Gura, Science 275: 751 (1997)).
The discovery of leptin and the characterization of the leptin receptor at the level of the central nervous system have opened a new route for the search for medicaments against obesity. This model, however, rapidly proved disappointing. Indeed, with only one exception (Montague et al., Nature 387:903 (1997)), the genes encoding leptin or its ob receptor have proved to be normal in obese human subjects. Furthermore and paradoxically, the plasma concentrations of leptin, the satiety hormone, are abnormally high in most obese human subjects.
Clearly there remains a need for novel medicaments that are useful for reducing body weight in humans. Such a pharmaceutical composition advantageously would help to control obesity and thereby alleviate many of the cardiovascular consequences associated with this condition.
One aspect of the present invention relates to an agent which influences the partitioning of dietary lipids between the liver and peripheral tissues for use as a medicament. In one embodiment, this agent can be used for treating a condition in which it is desirable to increase the partitioning of dietary lipids to the liver, reducing food intake in obese individuals, reducing the levels of free fatty acids in obese individuals, decreasing the body weight of obese individuals, or treating an obesity related condition selected from the group consisting of obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type II diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. According to another embodiment of the invention, the agent which influences the partitioning of dietary lipids between the liver and peripheral tissues is any one of: AdipoQ analogues, AdipoQ homologs, AdipoQ derivatives or fragments of any of the preceding agents. In yet another embodiment the agent includes an LSR antagonist or an LSR agonist.
Another aspect of the invention relates to a polypeptide that includes a consensus sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2 for use as a medicament.
Yet another aspect of the invention relates to a polypeptide comprising an amino acid sequence which alternatively may have at least 25% homology to one of the sequences of SEQ ID NOs.: 7-14, at least 50% homology to one of the sequences of SEQ ID NOs.: 7-14 or at least 80% homology to one of the sequences of SEQ ID NOs: 7-14 for use as a medicament.
Still yet another aspect of the invention relates to a C1q polypeptide, derivative, homologue or a fragment of any of the preceding compounds for use as a medicament.
A further aspect of the invention relates to an AdipoQ polypeptide or a derivative or homologue thereof or a fragment thereof for use as a medicament.
Another aspect of the invention relates to an ApM1 polypeptide or a derivative or homologue thereof or a fragment thereof for use as a medicament.
Still another aspect of the invention relates to the use of a compound that influences the partitioning of dietary lipids between the liver and peripheral tissues in the manufacture of a medicament for treating a condition in which the partitioning of dietary lipids to the liver is abnormal or higher than is desirable. In one embodiment, this medicament can be used for reducing food intake in obese individuals, reducing the levels of free fatty acids in obese individuals, decreasing the body weight of obese individuals, or treating an obesity related condition selected from the group consisting of obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type II diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. According to a different embodiment, the compound is one that is selected is any of: AdipoQ analogues, AdipoQ homologs, AdipoQ derivatives, and fragments of any of the preceding agents. According to yet a different embodiment the compound is an agonist or antagonist of the Lipolysis Stimulated Receptor. According to still another embodiment the compound can be any polypeptide comprising an amino acid sequence having at least 25% homology to one of the sequences of SEQ ID NOs.: 7-14, at least 50% homology to one of the sequences of SEQ ID NOs.: 7-14 or at least 80% homology to one of the sequences of SEQ ID NOs: 7-14. According to a different embodiment, the compound is a polypeptide that specifically binds a xcex3 subunit of the Lipolysis Stimulated Receptor or a gC1q-R or a gC1q-R homologue, but the compound is not a subunit of the lipolysis Stimulated Receptor. In this instance, the compound includes a polypeptide that can be C1q, AdipoQ, ApM1, Acrp 30, cerebellin or multimerin, or fragments of any of these polypeptides. In another embodiment, the compound that influences the partitioning of dietary lipids between the liver and peripheral tissues can be a polypeptide having binding specificity for a xcex3 subunit of the Lipolysis Stimulated Receptor or a gC1q-R or a gC1q-R homologue for the treatment of obesity. In this instance, the polypeptide is not a subunit of the Lipolysis Stimulated Receptor. According to another embodiment, the polypeptide can have about 25% homology to an ApM1 protein, about 50% homology to an ApM1 protein or about 80% homology to an ApM1 protein. More particularly, the polypeptide can be any of C1q, AdipoQ, ApM1, Acrp 30, cerebellin or multimerin, or fragments of any of these polypeptides. Additionally, the polypeptide can be a human polypeptide, and can be the ApM1 polypeptide or a fragment of the ApM1 polypeptide.
Another aspect of the invention relates to a polypeptide that specifically binds the gC1q-R protein for use in the treatment of obesity, wherein the polypeptide is not a subunit of the Lipolysis Stimulated Receptor. In one embodiment, the polypeptide can be any of C1q, AdipoQ, ApM1, Acrp 30, cerebellin or multimerin.
A still further aspect of the invention relates to a composition for modulating activity of the Lipolysis Stimulated Receptor. This composition includes a compound having binding specificity for the gC1q-R protein, but the compound cannot be a subunit of the Lipolysis Stimulated Receptor. The invented composition also includes a pharmaceutically acceptable carrier.
Another aspect of the invention relates to a composition for modulating activity of the Lipolysis Stimulated Receptor. This composition includes: (1) a polypeptide comprising an amino acid sequence at least 25% homologous to a sequence selected from the group consisting of any one of SEQ ID NOs 7-14, and a pharmaceutically acceptable carrier.
Still another aspect of the invention relates to a composition for modulating the activity of the Lipolysis Stimulated Receptor and includes: (1) a polypeptide that includes a consensus sequence that is either SEQ ID NO:1 or SEQ ID NO:2, and a pharmaceutically acceptable carrier.
Another aspect of the invention relates to a method of reducing plasma lipoprotein levels in an animal. This method includes the steps of: first identifying an animal having a measurable plasma lipoprotein level, then administering to the animal a composition that includes a pharmaceutically acceptable carrier and a polypeptide that is at least 25% homologous to an ApM1 protein and finally allowing passage of a period of time to permit reduction in the measurable plasma lipoprotein level. In a particular case the animal is a mammal. In a particular embodiment the composition may be administered by injection, for example by injecting intravenously. Alternatively, the composition may be administered by surgically implanting an infusion device that slowly releases the composition.
Another aspect of the invention relates to a method of identifying candidate pharmaceutical agents for reducing plasma triglyceride levels in an animal. This method involves first identifying a compound that includes a consensus sequence that may be either SEQ ID NO:1 or SEQ ID NO:2, obtaining a test animal having an initial level of plasma triglycerides, administering the compound to the test animal, waiting for a period of time, measuring a post-treatment level of plasma triglycerides in a blood sample obtained from the test animal and thereafter identifying as candidate pharmaceutical agents any compound that results in a post-treatment level of plasma triglycerides that is lower than the initial level. In one embodiment the test animal is a mammal and the method may involve feeding a high-fat meal to this mammal. The high-fat meal can include about 60% fat, about 20% protein, and about 20% carbohydrate. The fat component may include about 37% saturated fatty acids, about 36% polyunsaturated fatty acids and about 36% polyunsaturated fatly acids.
Still another aspect of the invention relates to a method for treating an animal having a condition in which it is desirable to increase the partitioning of dietary lipids to the liver. This method includes the step of administering an LSR agonist to the animal having the condition.
Still yet another aspect of the invention relates to a method for treating an animal having a condition in which it is desirable to decrease the partitioning of dietary lipids to the liver. This method includes the step of administering an LSR antagonist to the animal having the condition.
In another aspect, the invention comprises an agent which increases the activity of a compound which increases the partitioning of dietary lipids to the liver for use as a pharmaceutical. In one embodiment of this aspect, the agent is for use in reducing food intake in obese individuals, reducing the levels of free fatty acids in obese individuals, decreasing the body weight of obese individuals, or treating an obesity related condition selected from the group consisting of atherosclerosis (whether obesity-related or not), obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese subjects with Type II diabetes, and renal lesions caused by microangiopathy in obese subjects with Type II diabetes. In another embodiment of this aspect, the agent increases the activity of adipoQ, ApM1, a compound analogous to adipoQ or ApM1, or the LSR receptor. In a further embodiment of this aspect, the agent is selected from the group consisting of derivatives of adipoQ, ApM1, C1q, derivatives of a compound analogous to any of the preceding compounds wherein the derivatives exhibit greater activity than the corresponding wild type protein and antibodies capable of specifically binding the xcex3 subunit, the C1q receptor (gC1q-R) or a protein related thereto. In yet another embodiment of this aspect the agent is selected from the group consisting of derivatives of compounds comprising at least one of the sequences of SEQ ID NOs.: 1 and 2, derivatives of compounds comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, derivatives of compounds comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, and derivatives of compounds comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, wherein the derivatives exhibit greater activity than the corresponding wild type protein. In still a further embodiment of this aspect, the agent comprises a nucleic acid encoding a polypeptide or protein which influences the partitioning of dietary lipids between the liver and peripheral tissues for use as a medicament. In another embodiment of this aspect, the nucleic acid encodes a protein or polypeptide selected from the group consisting of adipoQ, ApM1, C1q, polypeptides analogous to ApM1, polypeptides having at least one of the consensus sequences of SEQ ID NO: 1 and SEQ ID NO: 2, analogs of any of the preceding polypeptides, homologs of any of the preceding polypeptides, derivatives of any of the preceding polypeptides, and fragments of any of the preceding polypeptides. In still another embodiment of this aspect, the nucleic acid encodes a polypeptide selected from the group consisting of polypeptides comprising an amino acid sequence having at least 25% homology to one of the sequences of SEQ ID NOs.: 7-14, polypeptides comprising an amino acid sequence having at least 50% homology to one of the sequences of SEQ ID NOs.: 7-14, and polypeptides comprising an amino acid sequence having at least 80% homology to one of the sequences of SEQ ID NOs: 7-14. In a further embodiment of this aspect, the agent is selected from the group consisting of small molecules and drugs. In yet another embodiment of this aspect, the agent is for administration to an individual having a below normal level of activity of adipoQ, ApM1, or an analoguous protein.
Another aspect of the present invention is an agent which decreases the activity of a compound which increases the partitioning of dietary lipids to the liver for use as a pharmaceutical. In one embodiment of this aspect, the agent is for use in treating cachexia in subjects with neoplastic or para-neoplastic syndrome or eating disorders. In another embodiment of this aspect, the agent decreases the activity of adipoQ, ApM1, a compound analogous to adipoQ or ApM1, or the LSR receptor. In a further embodiment of this aspect, the agent is an antibody which binds a compound selected from the group consisting of adipoQ, ApM1, C1q, a protein analogous to any of the preceding proteins, a derivative of adipoQ, C1qa, C1qb, C1qc, mul, cer, ApM1, or acrp which inhibits the activity of wild type adipoQ or wild type ApM1, fragments of any of the preceding polypeptides, the xcex3 subunit, the C1q receptor (gC1q-R) or a protein related thereto. In yet another embodiment of this aspect, the agent is an antibody which binds a polypeptide selected from the group consisting of polypeptides comprising at least one of the sequences of SEQ ID NOs.: 1 and 2, polypeptides comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, polypeptides comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, and polypeptides comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14. In a further embodiment of this aspect, the agent is selected from the group consisting of antisense nucleic acids to the adipoQ gene, the ApM1 gene or a portion thereof and nucleic acids capable of forming a triple helix with a portion of the adipoQ gene or the ApM1 gene. In yet another embodiment of this aspect, the agent is selected from the group consisting of antisense nucleic acids to a gene encoding a polypeptide comprising at least one of the sequences of SEQ ID NOs.: 1 and 2, a gene encoding a polypeptide comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, a gene encoding a polypeptide comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, and a gene encoding a polypeptide comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14. In a further embodiment of this aspect, the agent is selected from the group consisting of small molecules and drugs. In a further embodiment of this aspect, the agent is for administration to an individual having a level of adipoQ or ApM1 activity which is above normal.
Another aspect of the present invention is a method for determining whether an obese individual is at risk of suffering from a condition selected from the group consisting of a condition associated with a lower than desirable level of partioning of dietary lipids to the liver, obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese subjects with Type II diabetes, and renal lesions caused by microangiopathy in obese subjects with Type II diabetes, comprising the step of determining whether the individual has a lower than normal level of adipoQ activity, ApM1 activity, or activity of a compound analogous thereto.
Another aspect of the present invention is a method for increasing the partitioning of dietary lipids to the liver comprising administering an agent which increases the activity of a compound selected from the group consisting of adipoQ, ApM1, C1q, compounds analogous to C1q, compounds comprising at least one sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, compounds comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, compounds comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, and compounds comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14 to an individual. In one embodiment of this aspect, the individual suffers suffers from a condition selected from the group consisting of obesity, obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese subjects with Type II diabetes, and renal lesions caused by microangiopathy in obese subjects with Type II diabetes. In another embodiment of this aspect, the agent is selected from the group consisting of a derivative of adipoQ, ApM1 or an analogous compound which exhibits greater activity than the corresponding wild type protein, nucleic acids encoding adipoQ, ApM1, or an analogous compound, fragments of any of the preceding compounds, and nucleic acids encoding a derivative of adipoQ, ApM1, or an analogous compound having greater activity than the corresponding wild type protein, and fragments of any of the preceding compounds. In a further aspect of this embodiment, the agent is administered if it is determined that the level of ApM1, or an analogous protein in the individual is below normal.
Another aspect of the present invention is a method for decreasing the partitioning of dietary lipids to the liver comprising administering an agent which decreases the activity of a compound selected from the group consisting of adipoQ, ApM1, C1q, compounds analogous to C1q, compounds comprising at least one sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2, compounds comprising an amino acid sequence having at least 25% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, compounds comprising an amino acid sequence having at least 50% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14, and compounds comprising an amino acid sequence having at least 80% homology to a sequence selected from the group consisting of SEQ ID NOs. 7-14 to an individual. In one embodiment of this aspect, the individual suffers from a condition selected from the group consisting of cachexia in subjects with neoplastic or para-neoplastic syndrome or eating disorders. In another embodiment of this aspect, the agent is selected from the group consisting of an antibody which binds adipoQ, ApM1, C1q or an analogous protein, a derivative of adipoQ, C1qa, C1qb, C1qc, mul, cer, ApM1, or acrp which inhibits the activity of wild type adipoQ or wild type ApM1, a fragment of the derivative, antisense nucleic acids to the adipoQ gene, the ApM1 gene or a portion thereof, nucleic acids capable of forming a triple helix with a portion of the adipoQ gene or the ApM1 gene, and antibodies capable of binding the xcex3 subunit, the C1q receptor (gC1q-R) or a protein related thereto. In still another embodiment of this aspect, the agent is administered if it is determined that the level of adipoq, ApM1, or an analogous protein in the individual is above normal.
Another aspect of the present invention is a method of identifying a candidate compound for regulating the partitioning of dietary lipids between the liver and the adipose tissue comprising the steps of contacting the xcex3 subunit, the C1q receptor (gC1q-R) a protein related thereto, or a fragment thereof with one or more molecules to be tested for binding activity under conditions which permit specific binding of the molecule to the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof and determining whether the one or more molecules bind to the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof. In one embodiment of this aspect, the contacting step is performed using a cell expressing the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof. In another embodiment of this aspect, the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof is immobilized on a support. In yet another embodiment of this aspect, the method further comprises contacting the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof with a known ligand and determining the ability of the one or more molecules to be tested for binding activity to compete with the known ligand for binding to the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof. In a further embodiment of this aspect, the molecule to be tested for binding to the xcex3 subunit, C1q receptor (gC1q-R), protein related thereto, or fragment thereof is selected from the group consisting of polypeptides, peptides, derivatives or analogs thereof, drugs, and small molecules.
Another aspect of the invention relates to a method of identifying candidate pharmaceutical agents for reducing plasma triglyceride levels in an animal. This method involves first administering a compound to a test, and measuring a post-treatment level of plasma triglycerides in a blood sample obtained from the test animal. In one embodiment the test animal is a mammal and the method may involve feeding a high-fat meal to this mammal. The high-fat meal can include about 60% fat, about 20% protein, and about 20% carbohydrate. The fat component may include about 37% saturated fatty acids, about 36% polyunsaturated fatty acids and about 36% polyunsaturated fatty acids.